Restimulation of FVIII-specific memory responses by TLR agonists in the absence of FVIII in vitro and in vivo. (A-B) CD138⁻ spleen cells were obtained from hemophilic mice treated with 4 weekly doses of 200 ng of FVIII and restimulated in vitro with CpG-ODN (A) or ligands for TLRs 2, 3, 4, 5, and 7 (B) in the absence of FVIII (Loxo. = loxoribine). Newly formed anti-FVIII ASCs were detected by ELISPOT assay after 6 days of culture. Each spot represents one anti-FVIII ASC. Concentrations of CpG-ODN (A) and ligands for TLRs 2, 3, 4, 5, and 7 (B) are indicated. Representative ELISPOTs are presented. (C) CD138⁻ spleen cells were obtained from hemophilic mice treated with 4 weekly doses of 200 ng of FVIII and intravenously injected into naive hemophilic mice. One day after cell transfer, mice were injected with a single dose of PBS buffer (◇), PBS + 1000 μg of loxoribine (♦), 200 ng of FVIII (▵), or 200 ng of FVIII + 1000 μg of loxoribine (▴). TLR ligands were given intraperitoneally, PBS buffer and FVIII were given intravenously and blood samples were taken 3, 7, and 21 days after application, for the analysis of circulating anti-FVIII antibodies. Each point represents the results of an individual mouse. **P < .05.